Following stimulation, receptor and non-receptor tyrosine kinases initiate signaling mechanisms by phosphorylation of specific sets of cellular proteins. Such tyrosine phosphorylation can regulate enzyme activity (1, 2) or serve to recruit signaling proteins containing Src homology (SH2) ()or phosphotyrosine interacting domains(3, 4, 5) . In T-lymphocytes, a major phosphoprotein appearing in response to T-cell receptor cross-linking is the 120-kDa product of the c-cbl proto-oncogene(6, 7) . Cbl is located in cytosol and cytoskeleton and contains numerous proline-rich PXXP motifs, as well as several tyrosine residues that serve as possible binding sites for SH3 and SH2 domains, respectively(8) . The viral v-cbl oncogene product is a severely truncated, transforming form of c-Cbl found in the nucleus and cytosol(9) . We recently demonstrated that a population of Cbl is stably associated with the adaptor protein Grb2, and that activation of T-lymphocytes by cross-linking of the T-cell antigen receptor recruits PI 3-kinase into Cbl complexes(6) . These data suggest that Cbl may play an important role in facilitating signaling mechanisms in T-cells.

Although the possible role of Cbl in hematopoietic cell signaling has been emphasized previously(6, 7, 10) , the presence of Cbl in HeLa cells (9) prompted us to evaluate the role of this protein in other signaling systems. Of particular interest is the association of Cbl with Grb2, which is distributed widely among cell types. Grb2 is composed of one SH2 and two SH3 domains that link proteins containing phosphotyrosine to cellular proteins with proline-rich regions(11, 12) . For example, Grb2 is known to bind via its SH2 domain to phosphotyrosine residues in the EGF receptor (13, 14, 15) and to insulin receptor substrate 1 (IRS-1), which serves as a docking protein for SH2 domains in insulin-sensitive cells(16) . The NH-terminal SH3 domain binds stably to Cbl in hematopoietic cells(6, 7) , as well as to dynamin (17) and C3G (18) in other cell types. Grb2 also has a major role in the control of Ras signaling by binding to the guanine nucleotide-releasing factor Sos (19, 20, 21) . Both Sos and Cbl possess the identical proline-rich sequence PPVPPPR that has been identified as the region in Sos that binds to the Grb2 SH3 domain(19) . In this report we show that Cbl is indeed present in all cell types examined, and that addition of EGF to cultured human kidney cells causes the recruitment of Cbl into a complex containing the EGF receptor and its rapid tyrosine phosphorylation. Our data indicate that such association of Cbl with the EGF receptor may be mediated through Grb2.

EXPERIMENTAL PROCEDURES

Antibodies and Reagents

Cell Culture

Cell Stimulation and Preparation of Cell Lysates

Immunoprecipitation and Blotting

RESULTS AND DISCUSSION

We previously identified a major tyrosine-phosphorylated 120-kDa protein in T-cell receptor-activated Jurkat T-cells as the product of the Cbl proto-oncogene(6) . Although this protein has been found primarily in hematopoietic cells, Fig. 1shows that Cbl is present in Nonidet P-40 lysates from several other cells and tissues. In addition to Jurkat cells, high levels were found in two kidney cell lines (COS and 293), while CHO cells and 3T3-L1 preadipocytes expressed approximately 10% of this level. In differentiated 3T3-L1 adipocytes or rat adipose tissue, the concentration of Cbl was about 5% that of Jurkat cells. The low expression of Cbl in adipose cells is in agreement with a decrease of Cbl RNA during differentiation(24) . The two bands seen in 3T3-L1 lysates (right lane) represent Cbl with different stoichiometries of tyrosine phosphorylation, as defined by anti-phosphotyrosine blotting (not shown).

Figure 1: Distribution of the proto-oncogene product c-Cbl among various cell types. Lysates of cultured cells were prepared as described under ``Experimental Procedures.'' Proteins were solubilized in SDS-PAGE sample buffer, and 20 µg was loaded per lane. Following electrophoresis and transfer to nitrocellulose, proteins were blotted against Cbl antiserum and visualized by chemiluminescence.

The abundance of Cbl in 293 human kidney cells, coupled with the sensitivity of these cells to EGF(19) , led us to determine whether EGF receptor activation results in tyrosine phosphorylation of this protein. Fig. 2(lanes 1-5) reveals that stimulation of 293 cells with EGF for 2 min led to the appearance of a tyrosine-phosphorylated 120-kDa protein in total lysates (lane 2) and in anti-Cbl immunoprecipitates (lane 5) that blotted with Cbl antiserum (lower panel). EGF action also resulted in the appearance of other phosphotyrosine bands in anti-Cbl precipitates, most noticeably at 170 kDa. In Jurkat T-lymphocytes, cross-linking of the CD3/CD4 receptors (Fig. 2, lanes 6-10) increased tyrosine phosphorylation of Cbl and led to the co-precipitation of bands at 100, 83, 72, and 60 kDa, but not at 170 kDa, as detected with 4G10 antibody. As depicted in the lower panel of Fig. 2, the amount of Cbl that was immunoprecipitated with anti-Cbl antibody is similar in stimulated versus unstimulated 293 or Jurkat cells. These data demonstrate that EGF action causes marked tyrosine phosphorylation of Cbl in 293 cells. Moreover, EGF also enhances either the association of Cbl with tyrosine-phosphorylated proteins or the tyrosine phosphorylation of proteins constitutively bound to Cbl.

Figure 2: Cbl is tyrosine-phosphorylated upon activation of 293 cells with EGF. Jurkat and 293 cells were serum-starved for 4 h and activated either with EGF (62 ng/ml; lanes 1-5) or with OKT3 plus OKT4 mAbs (lanes 6-10) for 2 min at 37 °C. Lysates were precleared with normal rabbit serum before immunoprecipitation with normal (lanes 3 and 8) or anti-Cbl (lanes 4, 5, 9, and 10) serum. Lanes 1, 2, 6, and 7 are total lysates containing 20 µg of protein. After electrophoresis and transfer to nitrocellulose, samples were probed with 4G10 (top) or Cbl (bottom) antisera and visualized by chemiluminescence. Molecular mass markers (kDa) are denoted at right. The solid arrow points to the 170-kDa protein in lane 5.

The co-immunoprecipitation of a 170-kDa tyrosine-phosphorylated protein with Cbl in EGF-treated 293 cells but not activated Jurkat cells suggested that this protein might be the EGF receptor itself. Immunoprecipitation of 293 cell lysates with Cbl antiserum adsorbed a 170-kDa tyrosine-phosphorylated band (Fig. 3, upper panel, lane 8) that blotted faintly to EGF receptor antiserum from activated cells, but not with normal serum or from nonactivated cells (middle panel, lanes 5-8). The low signal may be due to the weaker recognition of tyrosine-phosphorylated receptors compared to nonphosphorylated receptors by the EGF receptor antibody on Western blots (compare lanes 4 and 5). EGF receptor immunoprecipitates showed a phosphotyrosine band at 120 kDa in activated (lane 5) but not in serum-starved cells (lane 4). Blotting with Cbl antibody confirmed that the 120-kDa band in EGF receptor immunoprecipitates is Cbl (lanes 3-6, lower panel). The absence of EGF receptor protein bound to anti-Cbl antibody and of Cbl protein in anti-EGF receptor immunoprecipitates of lysates of untreated 293 cells demonstrates EGF-mediated recruitment of Cbl to tyrosine-phosphorylated EGF receptors. Comparison by densitometry of the amount of the EGF receptor species in anti-Cbl immunoprecipitates versus EGF receptor immunoprecipitates indicates that approximately 30% of the total cellular EGF receptors associate with Cbl. This same value is obtained by analysis of either anti-EGF receptor or anti-tyrosine phosphorylation immunoblots (lanes 5 and 8). The large fraction of Cbl bound to the EGF receptor upon activation suggests an important functional relevance.

Figure 3: Activation of 293 cells with EGF induces the association of the EGF receptor with Grb2 and Cbl. Total lysates from serum-starved(-) or activated (+) cells are shown in lanes 1 and 2. Lysates were precleared with normal serum and immunoprecipitated with normal (lanes 3 and 6), anti-Cbl (lanes 7 and 8), or anti-EGF receptor antibody (lanes 4 and 5). Following electrophoresis and transfer to nitrocellulose, filters were probed with the antiphosphotyrosine antibody 4G10 (top), anti-EGF receptor, anti-Cbl, or anti-Grb2 (bottom), and visualized by chemiluminescence. Molecular mass markers (kDa) are shown at right.

In EGF-activated fibroblasts, a 170-kDa protein appearing in Grb2 and Sos immunoprecipitates has been identified as the EGF receptor(5, 19, 25, 26) , and it has been proposed that a complex of Sos, Grb2, and the EGF receptor is formed which leads to increased membrane localization and subsequent Ras activation. We therefore tested whether Grb2-Cbl complexes might be recruited to EGF receptors in an analogous manner. Blotting of the resolved proteins present in the anti-Cbl and anti-EGF receptor immunocomplexes shown in Fig. 3with Grb2 mAb (bottom panel) revealed that Grb2 was bound to the EGF receptor in cell lysates from EGF-activated cells (lane 5), but not from unstimulated cells (lane 4). Cbl immunoprecipitates, on the other hand, contained Grb2 in the basal state (lane 7), and EGF modestly increased the amount bound (lane 8). An increase in the Grb2/Cbl ratio was seen in several experiments (see Fig. 4). A constitutive association with Cbl via the NH-terminal SH3 domain of Grb2 has also been found in Jurkat cells(6, 7) . Taken together, the data in Fig. 3support the hypothesis that EGF receptor tyrosine phosphates recruit the SH2 domain of Grb2 proteins that are bound to Cbl through their NH-terminal SH3 domain. In addition, the activation-dependent increase of Grb2 complexed to Cbl supports our previous observation that the SH2 domain of Grb2 proteins can also bind directly to tyrosine-phosphorylated Cbl, despite the lack of a consensus YXNX motif(6, 15, 27) . It is not clear from our data whether a single Grb2 protein can bind Cbl with both its SH2 and SH3 domains or whether some Grb2 proteins bind through SH2 and some through their SH3 domain.

Figure 4: The EGF receptor and Grb2 are dissociated from Cbl by a proline-rich peptide. Cells were activated with EGF, and lysates reacted with Cbl antiserum, plus 0.05, 0.2, or 0.5 mM proline-rich peptide (lanes 4-6), at 4 °C for 2 h. Nitrocellulose filters containing electrophoresed proteins were blotted with anti-Tyr(P) (top), anti-Cbl (middle), or anti-Grb2 (bottom) antisera, and visualized by chemiluminescence.

In order to test more definitively whether the proline-rich regions of Cbl are required for binding Grb2 and the EGF receptor, peptide-mediated dissociation of Cbl-Grb2 and Cbl-EGF receptor complexes was attempted. A peptide containing the proline-rich sequence (PPVPPR) corresponding to amino acids 494-499 of Cbl was added to lysates from EGF-activated 293 cells, followed by immunoprecipitation with Cbl antiserum. Fig. 4shows that the EGF receptor (top panel) and Grb2 (bottom panel) were both dissociated from Cbl to the same extent as the peptide concentration was increased. A Western blot (Fig. 4, middle panel) confirms that the same amount of Cbl was immunoprecipitated at all peptide concentrations. Quantitation by densitometry of the relative amount of Grb2 and EGF receptor bound to Cbl in the presence of the proline-rich peptide (Fig. 5, closed symbols) revealed that the approximate half-maximal dissociation of the EGF receptor and Grb2 from Cbl occurs at 50 µM peptide. When a nonrelevant peptide was added (open symbols), no dissociation of Grb2 or EGF receptor from Cbl was observed. These results are consistent with EGF-induced coupling of the EGF receptor to Cbl via the SH2 and SH3 domains of Grb2. However, we cannot exclude the possibility that some other SH3-containing adaptor molecule may link the EGF receptor to Cbl in this system.

Figure 5: Dissociation of the EGF receptor and Grb2 from Cbl by proline-rich peptide. The autoradiograms depicted in Fig. 4were scanned by densitometry. Values are shown as a percentage of the density of Grb2 (squares) and EGF receptor (circles) in EGF-activated cells, incubated with either a proline-rich peptide (solid symbols) or a nonrelevant peptide (open symbols).

A simple hypothesis that explains the data presented here is that Grb2-Cbl complexes are recruited to tyrosine phosphorylation sites on the activated EGF receptor, and that tyrosine phosphorylation of Cbl results from its juxtaposition with the receptor kinase. This hypothesis predicts that Cbl may be recruited to complexes that include other proteins that bind the EGF receptor, such as Shc proteins. Shc SH2 and phosphotyrosine interacting domains bind EGF receptor tyrosine phosphates, and Shc itself becomes tyrosine-phosphorylated(3, 4, 20, 28) . Consistent with the above prediction, we observe Shc protein in immunoprecipitates of anti-Cbl antibody when lysates from EGF-treated 293 cells are used. ()Nonetheless, further experiments will be necessary to unequivocally test whether Cbl recruitment to EGF receptors is actually necessary for its tyrosine phosphorylation. Cells expressing mutant EGF receptors missing autophosphorylation sites fail to show a pronounced block in signaling (29) . Li et al. (29) have thus proposed that the EGF receptor may signal like the insulin receptor, in that SH2 domain-containing proteins are not directly bound, but rather receptor autophosphorylation causes the tyrosine phosphorylation of the docking proteins IRS-1 and Shc, which then serve as binding sites for SH2 proteins. For example, following insulin stimulation, Shc becomes rapidly tyrosine-phosphorylated, leading to Grb2-SH2 domain binding (30) . It will be important in future studies to test whether tyrosine phosphorylation of Cbl is normal in response to EGF receptors with ablated tyrosine phosphorylation sites.

The multiple potential tyrosine phosphorylation sites within the protein sequence of Cbl suggest it might serve to recruit multiple signaling proteins containing SH2 or phosphotyrosine interacting domains. In this sense, it may function similarly to IRS-1, which becomes tyrosine-phosphorylated at many sites in response to insulin and binds SH2 domain-containing proteins(16) . Upon growth factor stimulation, both proteins become transiently associated with higher concentrations of SH3 and SH2 domain proteins, including Grb2, Nck, and PI 3-kinase(6, 7, 10, 16) . Interestingly, we observe several tyrosine-phosphorylated proteins that co-immunoprecipitated with Cbl in both Jurkat and 293 cells (Fig. 2, lanes 5, 9, and 10). One of these appears to be the tyrosine kinase Fyn, which binds in vitro to a 120-kDa tyrosine-phosphorylated protein through SH2 and SH3 domains (31, 32) . Evidence suggests that the formation of transient complexes of Cbl with SH2 domain proteins regulates cell growth(9) . This may be achieved by allowing localized increases in the activity of those proteins that bind via SH2-phosphotyrosine interaction, analogous to PI 3-kinase and Syp phosphatase interactions with phosphopeptides(14, 33) . Such localized increases in specific activity may be sufficient to promote signaling pathways. In support of this, we have noted a 4-fold increase in PI 3-kinase bound to Cbl upon T-cell receptor cross-linking in Jurkat T-cells(6) . It is not clear whether PI 3-kinase is recruited to Cbl in 293 cells because activity of this enzyme is very low in this model system(34) . In any case, the findings presented here showing recruitment of Cbl to EGF receptor complexes and its marked tyrosine phosphorylation in response to EGF suggest that Cbl may play a key role in EGF receptor signaling.

FOOTNOTES

*

This work was supported by the Juvenile Diabetes Foundation International Program Project Grant 892004 and the Peptide Synthesis Facility at the University of Massachusetts Medical School Diabetes and Endocrinology Research Center through National Institutes of Health Grant P30DK32520. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 508-856-6929; Fax: 508-856-4289.

()

The abbreviations used are: SH, Src homology; EGF, epidermal growth factor; IRS-1, insulin receptor substrate 1; mAb, monoclonal antibody; PI 3-kinase, phosphatidylinositol 3-kinase; Sos, son-of-sevenless.

()

H. Meisner and M. P. Czech, unpublished data.

ACKNOWLEDGEMENTS

Note Added in Proof-After the preparation of this report, two laboratories have demonstrated that Cbl becomes tyrosine-phosphorylated by EGF in fibroblasts overexpressing the EGF receptor(35, 36) . Galisteo et al. (36) conclude that Cbl is not complexed to Grb2 and binds directly to the EGF receptor in HER14 cells. The results presented here and elsewhere (6, 7) document a stable Grb2-Cbl complex in Jurkat and 293 cells, suggesting that two mechanisms may operate to couple EGF receptors to Cbl.

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				J. Chu, Y. Liu, G. A. Koretzky, and D. L. Durden SLP-76-Cbl-Grb2-Shc Interactions in Fcgamma RI Signaling Blood, September 1, 1998; 92(5): 1697 - 1706. [Abstract] [Full Text] [PDF]

				R. K. Park, W. T. Kyono, Y. Liu, and D. L. Durden CBL-GRB2 Interaction in Myeloid Immunoreceptor Tyrosine Activation Motif Signaling J. Immunol., May 15, 1998; 160(10): 5018 - 5027. [Abstract] [Full Text] [PDF]

				E. A. Feshchenko, W. Y. Langdon, and A. Y. Tsygankov Fyn, Yes, and Syk Phosphorylation Sites in c-Cbl Map to the Same Tyrosine Residues That Become Phosphorylated in Activated T Cells J. Biol. Chem., April 3, 1998; 273(14): 8323 - 8331. [Abstract] [Full Text] [PDF]

				B. L. Rellahan, L. J. Graham, B. Stoica, K. E. DeBell, and E. Bonvini Cbl-mediated Regulation of T Cell Receptor-induced AP1 Activation. IMPLICATIONS FOR ACTIVATION VIA THE Ras SIGNALING PATHWAY J. Biol. Chem., December 5, 1997; 272(49): 30806 - 30811. [Abstract] [Full Text] [PDF]

				M. Ishiki, T. Sasaoka, H. Ishihara, T. Imamura, I. Usui, Y. Takata, and M. Kobayashi Evidence for Functional Roles of Crk-II in Insulin and Epidermal Growth Factor Signaling in Rat-1 Fibroblasts Overexpressing Insulin Receptors Endocrinology, November 1, 1997; 138(11): 4950 - 4958. [Abstract] [Full Text] [PDF]

				E. D. Fixman, M. Holgado-Madruga, L. Nguyen, D. M. Kamikura, T. M. Fournier, A. J. Wong, and M. Park Efficient Cellular Transformation by the Met Oncoprotein Requires a Functional Grb2 Binding Site and Correlates with Phosphorylation of the Grb2-associated Proteins, Cbl and Gab1 J. Biol. Chem., August 8, 1997; 272(32): 20167 - 20172. [Abstract] [Full Text] [PDF]

				D. L. Barber, J. M. Mason, T. Fukazawa, K. A. Reedquist, B. J. Druker, H. Band, and A. D. D'Andrea Erythropoietin and Interleukin-3 Activate Tyrosine Phosphorylation of CBL and Association With CRK Adaptor Proteins Blood, May 1, 1997; 89(9): 3166 - 3174. [Abstract] [Full Text] [PDF]

				H. Odai, K. Sasaki, A. Iwamatsu, T. Nakamoto, H. Ueno, T. Yamagata, K. Mitani, Y. Yazaki, and H. Hirai Purification and Molecular Cloning of SH2- and SH3-Containing Inositol Polyphosphate-5-Phosphatase, Which Is Involved in the Signaling Pathway of Granulocyte-Macrophage Colony-Stimulating Factor, Erythropoietin, and Bcr-Abl Blood, April 15, 1997; 89(8): 2745 - 2756. [Abstract] [Full Text] [PDF]

				M. Sattler, R. Salgia, G. Shrikhande, S. Verma, E. Pisick, K. V.S. Prasad, and J. D. Griffin Steel Factor Induces Tyrosine Phosphorylation of CRKL and Binding of CRKL to a Complex Containing c-Kit, Phosphatidylinositol 3-Kinase, and p120CBL J. Biol. Chem., April 11, 1997; 272(15): 10248 - 10253. [Abstract] [Full Text] [PDF]

				H. Ueno, K. Sasaki, K. Miyagawa, H. Honda, K. Mitani, Y. Yazaki, and H. Hirai Antisense Repression of Proto-oncogene c-Cbl Enhances Activation of the JAK-STAT Pathway but Not the Ras Pathway in Epidermal Growth Factor Receptor Signaling J. Biol. Chem., March 28, 1997; 272(13): 8739 - 8743. [Abstract] [Full Text] [PDF]

				Y.-C. Liu, C. Elly, W. Y. Langdon, and A. Altman Ras-dependent, Ca2+-stimulated Activation of Nuclear Factor of Activated T Cells by a Constitutively Active Cbl Mutant in T Cells J. Biol. Chem., January 3, 1997; 272(1): 168 - 173. [Abstract] [Full Text] [PDF]

				JeraldA. Donovan, Y. Ota, WallaceY. Langdon, and LawrenceE. Samelson Regulation of the Association of p120cbl with Grb2 in Jurkat T Cells J. Biol. Chem., October 18, 1996; 271(42): 26369 - 26374. [Abstract] [Full Text] [PDF]